Geraniol-evoked electro-olfactogram recordings from olfactory epithelia show significant differences in adaptation for wild-type compared to eNOS-deficient mice.

<p><i>A,</i> Representative recordings of odor-responses to repetitive application of geraniol (1 second duration; 4 seconds interstimulus interval) in wild-type (black) and eNOS deficient mice (red). <i>B,</i> Histogram of mean amplitudes of consecutive responses normalized to the amplitude of the first geraniol response in a stimulus series of wild-type (black bars) and eNOS deficient mice (red). <i>C</i>, Histogram of mean amplitudes of consecutive responses normalized to the amplitude of the first geraniol response in a stimulus series of wild-type olfactory epithelium incubated with Ringer's solution (dark grey bars) or L-NMMA (100 µM, light grey bars). Please notice that incubation of the OE with Ringer`s solution or drug in general led to slower adaptation kinetics, but comparison of the two conditions still shows a significant difference. <i>D,</i> Representative recordings of odor responses to geraniol pulses (2 seconds duration; 28 seconds interstimulus interval). <i>E,</i> Mean amplitude of the second geraniol response normalized to the amplitude of the first response.</p

Amperometric NO-recordings of individual OSNs of wild-type, OMP-GFP, or eNOS-deficient mutant mice.

<p><i>A,</i> Application of KCl (45 mM) induces a robust NO-signal in an isolated wild-type OSNs. <i>B,</i> Veratridine (10 µM) induces a NO-signal in a wild-type OSN, but fails to induce NO release in an OSN of an eNOS-deficient mutant mouse (eNOS-delMu). <i>C,</i> Stimulation of the olfactory signal transduction pathway with forskolin (20 µM) led to a clear NO-signal in OSNs of OMP-GFP mice. Signals could be prevented by pre-incubation with L-NAME (1 mM) or by performing the experiments in extracellular medium with low Ca²⁺-concentration (1 nM). <i>D,</i> Application of the odorants geraniol (200 µM) or octanal (500 µM) produced NO-signals in OMP-GFP mice. Arrows mark the application of the indicated stimulus. Large deflections of the signal at the time of application represented artefacts that occurred in some experiments (forskolin, geraniol). These artefacts were blanked for clarity.</p

Wild-type and eNOS-deletion mutant mice show no differences in amplitude and in decay kinetics of responses to single pulses of geraniol (1 s duration).

<p>(A) Absolute (open squares) and mean amplitudes (filled diamonds) of geraniol responses. Mean values were 1.82±0.17 mV for wild-type and 1.87±0.27 mV for eNOS deletion mutants. (B) Decay kinetics measured as curve width at 50% of the response amplitude. Mean values were 2.49±0.13 s for wild-type and 2.52±0.19 s for eNOS deletion mutants. Error bars indicate SEM.</p

mRNA of the endothelial isoform of NO-synthase (eNOS) is expressed in olfactory sensory neurons.

<p><i>A,</i> RT-PCR analysis of 1500 purified OSNs with primers specific for eNOS and Gα_olf. <i>B,</i><i> In situ</i> hybridization of eNOS-specific anti-sense and sense probes to cryosections of the murine olfactory epithelium. The scale bars represent 20 µm.</p

Quantification of BrdU-labeled basal cells in the olfactory epithelium of wild-type and eNOS deficient mice shows no difference in numbers of proliferating cells.

<p>(A) Schematic drawing of a mouse head with the three coronal section levels (referred to as 1, 2 and 3) that were used for quantification of BrdU positive cells. Scheme adapted from A. Puche (<a href="http://www.apuche.org" target="_blank">www.apuche.org</a>). (B) Reconstructions of coronal sections from these section levels and their respective localization (1, 2 and 3). (C) Exemplary anti-BrdU immunofluorescence from the nasal septum of wild-type (WT) and deletion mutant (delMu) mice. Dashed lines indicate the olfactory epithelium. Arrows represent 100 µm. (D) Cell densities of BrdU positive cells of the nasal septum of WT and delMu animals for all three section levels. Error bars indicate SEM.</p

eNOS-protein is localized to somata, dendrites and olfactory knobs but not to the cilia of mature OSNs.

<p><i>A,</i> eNOS immunostaining of cryosections of the olfactory epithelium of OMP-GFP mice. Pictures show endogenous GFP-fluorescence of OSNs (green), eNOS-specific immunostaining (red) as well as the overlay of the endogenous fluorescence and the immunostaining. <i>B,</i> Control without primary antibody. <i>C,</i> Double immunostaining of GFP-positive OSNs with antibodies for eNOS and adenylyl cyclase type III. Pictures show endogenous GFP-fluorescence of OSNs (green), eNOS-specific immunofluorescence (red), adenylyl cyclase type III (blue) and the overlay. The scale bars represent 20 µm.</p